Characterization of neural stemness status through the neurogenesis process for bone marrow mesenchymal stem cells

Figures & data

Figure 1 Morphology characteristics of mouse MSCs cultured in MEM +20% FBS after 24 hours as revealed under inverted microscope.

Notes: (A) 10 x; (B) 20x.
Abbreviations: MSCs, mesenchymal stem cells; MEM, minimum essential medium; FBS, fetal bovine serum.

Figure 2 After 72 hours of culturing mouse MSCs in MEM +20% FBS, note that some of the cells became adherent and began to elongate as revealed under inverted microscope.

Notes: (A)10×; (B)20×.
Abbreviations: MSCs, mesenchymal stem cells; MEM, minimum essential medium; FBS, fetal bovine serum.

Figure 3 Maintenance of MSCs in culture.

Notes: (A and B) After 5–6 days of culturing (monolayer cells as P0), note that the colonies interconnected with each other and reached a confluent stage. (C and D) after the first passage (P1) culture of MSCs. As revealed under inverted microscope for 10× and 20× for each passage, respectively.
Abbreviation: MSCs, mesenchymal stem cells.

Figure 4 Immunophenotypic analysis of MSCs at the first passage revealed under light microscope show that the MSCs were negative by cell stained with blue color.

Notes: (A) 10×; (B) 40×.
Abbreviation: MSCs, mesenchymal stem cells.

Figure 5 Immunophenotypic analysis of MSCs at the first passage revealed under light microscope show that the MSCs were positive cells stained with brown color.

Notes: (A and B) CD44, (C and D) CD90, (E and F) CD105, note that all CDs are shown at 10× and 40×, respectively.
Abbreviation: MSCs, mesenchymal stem cells.

Figure 6 MSCs after induced differentiation by BME, which showed the spherical shape of cells and their branched form toward the neural cells as revealed under inverted microscope.

Notes: All figures showed in 10×. The panels (A–E) were presented in 24–34 hours exposure times to differentiation media.
Abbreviations: MSCs, mesenchymal stem cells; BME, β-mercaptoethanol.

Figure 7 ICC detection results of MSCs after induced differentiation by BME, which showed the increase of NES and NF-L protein through the different exposure times, compared with no increase of MAP-2 protein as revealed under light microscope.

Notes: All figures showed in 10×. The panels (A–E) presented (MSC-29 hours) the exposure times of each three markers.
Abbreviations: ICC, immunocytochemistry; MSCs, mesenchymal stem cells; BME, β-mercaptoethanol; NES, nestin, NF-L, neurofilament light; MAP-2, microtubule-associated protein 2.

Figure 8 The protein expression levels of ICC assay for NES, NF-L, and MAP-2 antibodies of neural cells after being induced by BME.

Notes: Note that this data were significant at P<0.05 and represent as mean ± SE.
Abbreviations: ICC, immunocytochemistry; NES, nestin; NF-L, neurofilament light; MAP-2, microtubule-associated protein 2; BME, β-mercaptoethanol; MSCs, mesenchymal stem cells; se, standard error.

Figure 9 mRNA expression levels of the real time-PCR analysis of NES, NF-L, and MAP-2 genes in MSCs treated with BME.

Notes: There were high expression levels with significant difference in the NES gene in the first 24 hours, with low expression of each NF-L gene, then decreased in NES gene levels and increased in NF-L compared with control MSCs (P<0.05). Moreover, there was a decrease in the expression levels of MAP-2, with significant difference at different exposure times (P<0.05). Note that all of these three primers were normalized with the GAPDH gene (A) Amplification plot of all primers, (B) dissociation curve of all primers, (C) results of real time-PCR. Note that these data were significant at P>0.05 and represent the ∆CT of mean ± SEM.
Abbreviations: PCR, polymerase chain reaction; NES, nestin; NF-L, neurofilament light; MAP-2, microtubule-associated protein 2; MSCs, mesenchymal stem cells; BME, β-mercaptoethanol; SEM, standard error of mean.

Figure 10 Neural stem cells induction by (A) 6 and (B) 24 hours exposure to BME as revealed under inverted microscope.

Note: All figures showed in 10×.
Abbreviations: BME, β-mercaptoethanol; MSCs, mesenchymal stem cells.

Figure 11 Neural stem cells induction after 24 hours exposure to BME using four different culturing media formula.

Notes: From A–C for each formula, (A) 24 hours exposure to BME, (B) Passage 1, (C) Passage 2, as revealed under inverted microscope. Note that all figures are shown in 10×.
Abbreviations: BME, β-mercaptoethanol; NSCs, neural stem cells.

Figure 12 RT-PCR analysis of NES, NF-L, and MAP-2 genes in NSCs using BME, which indicated the stability of the NES gene in all the different culturing media formulas and the instability of each NF-L and MAP-2 genes compared with the 24 hours exposure (the production stage of NSCs).

Notes: GAPDH was used as control (housekeeping gene). (A) Amplification plot of all primers, (B) dissociation curve of all primers, (C and D) results of RT-PCR for 6 and 24 hours, respectively. Note that these data were significant at P<0.05 and represent the ∆CT of mean ± seM.
Abbreviations: RT-PCR, real time-PCR; NES, nestin; NF-L, neurofilament light; MAP-2, microtubule-associated protein 2; NSCs, neural stem cells; BME, β-mercaptoethanol; SEM, standard error of mean; EGF, epidermal growth factor; FGF, fibroblast growth factor; MSCs, mesenchymal stem cells.

Figure 13 Showing the neurogenesis pathway that MSCs induced toward neurons, where we can indicate the neural stemness state by following gene expression of the neurogenesis markers (NES, NF-l, and MAP-2) after prove of stemness at 6 hours of exposure to BME.

Abbreviations: MSCs, mesenchymal stem cells; NES, nestin; NF-L, neurofilament light; MAP-2, microtubule-associated protein 2; BME, β-mercaptoethanol.